Ca2+ signaling regulate fluid and electrolyte secretion by salivary gland (SG) cells. This polarized function of SG dictates polarized organization and functioning of Ca2+ signaling complexes in cellular microdomains. Scaffolding proteins plays critical roles in the assembly AND regulation of Ca2+ signaling proteins within the complexes. A central component of the Ca2+ signaling complexes is Ca2+ influx, which is mediated by TRPC channels. TRPC3 and TRPC6 are the dominant channels in SG. How the scaffolds Spinophilin (SPL), Neurabin (NRB) and Homerl regulates the action of GPCRs and TRPC3/6 channels is the theme of this proposal. Towards achieving our goals we found the regulation of Ca2+ signaling by the SPL/NRB pair, the role of Homerl in trafficking of TRPC channels, regulation of TRPC6 activity by SPL and by the newly discovered STIM1. These findings led to development of the following specific aims to probe the role of scaffolds in SG Ca2+ signaling. 1. Determine the role of SPL/NRB in regulating GPCRs Ca2+ signaling by RGS proteins in SG cells. This will be achieved by a) Identifying the NRB domain that binds RGS proteins and the relationship between SPL and NRB binding of RGS proteins, b) Determining the role of NRB in Ca2+ signaling in RGS2-/- and NRB-/- cells and c) Characterizing Ca2+ signaling in SG cells from SPL-/- and NRB-/- mice. 2. Explore the role of Homerl in TRPC channels translocation and Ca2+ influx by: a) studying translocation and retrieval of TRPC3/6 in SG cells and the role of store depletion and Homerl in both activities;b) Extend the findings in native cells by studying translocation of TRPC3/6-YFP expressed in HEK cells by biotinylation and TIRF assays. 3. Study regulation of TRPC3/6 by SPL/NRB by: a) Identifying the two SPL/NRB domains that interact with TRPC3/6;b) determine the effect of SPL/NRB in TRPC3/6 translocation and retrieval;c) characterize the regulation of TRPC3/6 channel activity by SPL/NRB in vivo using SPL-/- and NRB-/- cells. 4. Study regulation of TRPC3/6 by STIM1 by: a) determine the regulation of NATIVE and expressed TRP3/6 channels by STIM1, b) explore the mechanism by which STIM1 regulates TRPC3/6. The proposed work explores novel regulatory mechanisms in Ca2+ signaling and their relevance to regulation of SG fluid and electrolyte secretion.